1. Field of the Invention
The invention relates to laser spectroscopy and, in particular, relates to laser fluorometric detectors for microcolumn chromatography, and can be used for detecting ultratrace amounts of fluorescent liquids.
2. Description of the Prior Art
The following requirements are usually the goals in the development of LC detectors: high sensitivity, low detection limit, low noise level, and broad linearity range. In addition, chromatographic peak resolution also imposes certain limitations on the design of the cell. It should have a limited volume, the isolated components should not mix, and the detector channel should be capable of being washed very fast.
The concentrational sensitivity of the detector is proportional to the amount of the substance in the measuring cell. To leave the detector resolution unaffected, the following condition should be observed: ##EQU1## where V.sub.o is the detected volume including the cell volume and the volume of the detector channel from the outlet of the chromatographic column to the measuring cell;
V.sub.1 is the volume of the effluent in a chromatographic peak; PA1 S is the ID area of the column; and PA1 1 is the column length.
On the other hand, if V.sub.o &lt;&lt;V.sub.1, the detector sensitivity drops, since the amount of the substance in the cell becomes less. Each chromatographic column has, therefore, its own optimal volume of detection, which should be selected.
Known in the art is a laser fluorometric detector for microcolumn chromatography (cf., for example, L.W. Hershberger, J.B. Callis, and G.D. Christian, Analytical Chemistry, Vol. 51, No. 9, August 1979, pp. 1444-1446), which comprises several optically connected components: a laser, a unit for forming laser emission, a flow-through cell designed for communication with the chromatographic column, and a receiver of fluorescence emission.
The flow-through cell of this detector is a chamber with quartz windows on each of the four sides. The internal volume of the chamber is filled with the efluent which is also used in the flow channel of the chromatographic column. The lower part of the chamber is fitted with a hydrodynamic jet nozzle through which the sample is injected. The fluorescence emission is collected by the objective of the receiver in a direction perpendicular to the excitation radiation.
The disadvantage of this detector is its insufficient sensitivity.
This is due to the fact that the detected volume of the effluent is substantially smaller in relation to the volume of the chromatographic peaks, since the cross-section of the stream flowing through the cell is substantially less than the cross-section of the internal diameter of the chromatographic column. One more contributing factor consists in that the sample stream is surrounded by the effluent stream whose thickness exceeds the sample stream diameter by a factor of 10 to 20. Fluorescence of the effluent, which adds to the noise level, is a serious handicap to the useful signal recording.
Also known in the art is a laser fluorometric detector for microcolumn chromatography (cf., for example, H.Todoriki, A.Hirakawa, Chemical & Pharmaceutical Bulletin, Vol. 28, No. 4, 1980, pp. 1337-1339), comprising the following optically connected components: a laser, a laser emission forming unit, a flow-through cell intended for communication with the chromatographic column, a diaphragm for adjusting the sample volume in the cell, and a fluorescence emission receiver.
The flow-through cell in this device is a quartz tube with another quartz tube, of a lesser diameter, connected perpendicular thereto as a sample inlet. An optical fiber is introduced into the upper end of the flow cell, a micro-lens being attached to the exit end of the fiber, which is the laser emission forming unit.
The micro-lens is placed somewhat above the region where the two tubes are connected in order to reduce the divergence of the laser beam after the fiber. The fluorescence signal is produced in the zone where the flowing sample enters the large-diameter tube from the smaller-diameter tube. The fiber with the micro-lens is placed so that the diverging laser beam does not reach the tube walls in the zone where the fluorescence emission is collected. This zone can be selected by means of a round adjustable diaphragm installed on one axis with the sample inlet tube. In this manner the photoreceiver is protected from the scattered laser light.
This device is deficient in that the resolution of the detector is not high enough. Chromatographic fractions are seriously disturbed when the sample flow turns from the smaller diameter tube into the larger diameter tube, and the laminar nature of the stream is upset.
In addition, in the two above detectors fluorescence was excited by an unparallel laser beam, since the latter is focused by spherical lenses. This, in turn, adds to the scattered laser radiation on the boundaries where media having different refractive indices meet.
Also, the above detectors do not provide for optimal selection of sample volumes for different microcolumns. In case the microcolumn is replaced by another, the sensitivity or resolution of detection can be affected.